ABSTRACT Project 1 investigates ??TCR function spanning in vitro single molecule and single cell assays that interrogate force-dependent mechanosensor activation by peptide/MHC (pMHC) ligands with optical tweezers to in vivo assays with corresponding retrogenic ?? T cells addressing biological consequences such as nave T cell expansion and memory T cell formation and maintenance. Thus, Project 1 couples ??TCR molecular mechanism with functional biology. From a systems perspective, Project 1 links with Project 2 of the preTCR (pT??) by executing parallel tests on the mature ??TCR. Parameters will also be measured for single cell activation including the activation threshold, force range for activation and intracellular calcium profile. Project 1 is tightly integrated with NMR and Project 3, where structural and conformational changes as a consequence of mutations (strengthening, weakening, substitutions) will be defined and mapped onto changes in measured parameters. At the single molecule level, conformational change(s), force bond lifetime, extension magnitude, critical force and transition energy landscape will be measured. At the single cell level, the physical and chemical requirements to trigger (critical force window and pMHC density) will be characterized. TCRs will also be evaluated in vivo (nave mouse) for their ability to induce T cell proliferation upon exposure to infectious pathogens such as influenza A and their ability to form central or tissue resident memory T cells. Single cells will be triggered at critical force thresholds and then recovered through micropipette aspiration for transcriptome analysis. The overarching goal is to pair single molecule and single cell measurements of Aim 1 with functional biological impact of Aim 2. Strategically, the project will execute a cycle of measurements involving single molecule single cell, functional biology, structure biology utilizing NMR and molecular dynamics to permit refinement of our models and confirmation or rejection of our atomistic hypotheses to explain ??TCR mechanobiology.